Polar Growth in Corynebacterium glutamicum Has a Flexible Cell Wall Synthase Requirement.

Members of the Corynebacterineae suborder of bacteria, including major pathogens such as Mycobacterium tuberculosis, grow via the insertion of new cell wall peptidoglycan (PG) material at their poles. This mode of elongation differs from that used by Escherichia coli and other more well-studied model organisms that grow by inserting new PG at dispersed sites along their cell body. Dispersed cell elongation is known to strictly require the SEDS-type PG synthase called RodA, whereas the other major class of PG synthases called class A penicillin-binding proteins (aPBPs) are not required for this mode of growth. Instead, they are thought to be important for maintaining the integrity of the PG matrix in organisms growing by dispersed elongation. In contrast, based on prior genetic studies in M. tuberculosis and related members of the Corynebacterineae suborder, the aPBPs are widely believed to be essential for polar growth, with RodA being dispensable. However, polar growth has not been directly assessed in mycobacterial or corynebacterial mutants lacking aPBP-type PG synthases. We therefore investigated the relative roles of aPBPs and RodA in polar growth using Corynebacterium glutamicum as a model member of Corynebacterineae. Notably, we discovered that the aPBPs are dispensable for polar growth and that this growth mode can be mediated by either an aPBP-type or a SEDS-type enzyme functioning as the sole elongation PG synthase. Thus, our results reveal that the mechanism of polar elongation is fundamentally flexible and, unlike dispersed elongation, can be effectively mediated in C. glutamicum by either a SEDS-bPBP or an aPBP-type synthase. IMPORTANCE The Corynebacterineae suborder includes a number of major bacterial pathogens. These organisms grow by polar extension unlike most well-studied model bacteria, which grow by inserting wall material at dispersed sites along their length. A better understanding of polar growth promises to uncover new avenues for targeting mycobacterial and corynebacterial infections. Here, we investigated the roles of the different classes of cell wall synthases for polar growth using Corynebacterium glutamicum as a model. We discovered that the polar growth mechanism is surprisingly flexible in this organism and, unlike dispersed synthesis, can function using either of the two known types of cell wall synthase enzymes.

Effect of low-level ultrasound treatment on the production of L-leucine by Corynebacterium glutamicum in fed-batch culture.

Various process intensification methods were proposed to improve the yield, quality, and safety of fermented products. Here, we report the enhancement of L-leucine production by Corynebacterium glutamicum CP using ultrasound-assisted fed-batch fermentation. Response surface methodology was employed to optimize the sonication conditions. At an ultrasonic power density of 94 W/L, frequency of 25 kHz, interval of 31 min, and duration of 37 s, C. glutamicum CP produced 52.89 g/L of L-leucine in 44 h, representing a 21.6% increase compared with the control. The production performance of L-leucine was also improved under ultrasonic treatment. Moreover, the effects of ultrasound treatment on the fermentation performance of L-leucine were studied in terms of cell morphology, cell membrane permeability, and enzyme activity. The results indicate that ultrasonication is an efficient method for the intensification of L-leucine production by C. glutamicum CP.

Conversion Efficiencies of a Few Living Microbial Cells Detected at a High Throughput by Droplet-Based ESI-MS.

The label-free and sensitive detection of synthesis products from single microbial cells remains the bottleneck for determining the specific turnover numbers of individual whole-cell biocatalysts. We demonstrate the detection of lysine synthesized by only a few living cells in microfluidic droplets via mass spectrometry. Biocatalyst turnover numbers were analyzed using rationally designed reaction environments compatible with mass spectrometry, which were decoupled from cell growth and showed high specific turnover rates (∼1 fmol/(cell h)), high conversion yields (25%), and long-term catalyst stability (>14h). The heterogeneity of the cellular reactivity of only 15 ± 5 single biocatalysts per droplet could be demonstrated for the first time by parallelizing the droplet incubation. These results enable the resolution of biocatalysis beyond averages of populations. This is a key step toward quantifying specific reactivities of single cells as minimal functional catalytic units.

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum.

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates.

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

Corynebacterium glutamicum whiA plays roles in cell division, cell envelope formation, and general cell physiology.

The whiA gene is widely distributed among Gram-positive bacteria. Although the encoded protein has conserved N-terminal homing endonuclease scaffold and C-terminal helix-turn-helix DNA-binding domains, whiA plays a unique physiological role in its host organisms, reflecting a long history of evolution. Here, we used genetic approaches to unveil the physiological function of whiA in Corynebacterium glutamicum. We found that cells lacking whiA (ΔwhiA) were unable to grow in minimal medium containing glucose, although reduced growth was observed in complex medium. The ΔwhiA strain showed altered transcription of the cell division genes ftsZ, sepF, ftsK, crgA, divIVA, and amiC genes. Accordingly, ΔwhiA cells exhibited large, elongated, branched, and bud-shaped morphologies. In addition, some genes, including fas-IA, fas-IB, accD1, and cmrA, which help synthesize the fatty acid and cell envelope component mycolic acid, showed altered transcription in the ΔwhiA strain. Further, treS, treY, otsA, and otsB, which are involved in the biosynthesis of the outer envelope component trehalose, were down-regulated in the ΔwhiA strain. 2D-PAGE analysis of the ΔwhiA mutant showed that proteins involved in other cellular activities were also affected by the loss of whiA. These findings suggest that C. glutamicum whiA plays a critical role in cell division, envelope formation, and general cell physiology.

The conserved actinobacterial transcriptional regulator FtsR controls expression of ftsZ and further target genes and influences growth and cell division in Corynebacterium glutamicum.

BACKGROUND: Key mechanisms of cell division and its regulation are well understood in model bacteria such as Escherichia coli and Bacillus subtilis. In contrast, current knowledge on the regulation of cell division in Actinobacteria is rather limited. FtsZ is one of the key players in this process, but nothing is known about its transcriptional regulation in Corynebacterium glutamicum, a model organism of the Corynebacteriales. RESULTS: In this study, we used DNA affinity chromatography to search for transcriptional regulators of ftsZ in C. glutamicum and identified the Cg1631 protein as candidate, which was named FtsR. Both deletion and overexpression of ftsR caused growth defects and an altered cell morphology. Plasmid-based expression of native ftsR or of homologs of the pathogenic relatives Corynebacterium diphtheriae and Mycobacterium tuberculosis in the ΔftsR mutant could at least partially reverse the mutant phenotype. Absence of ftsR caused decreased expression of ftsZ, in line with an activator function of FtsR. In vivo crosslinking followed by affinity purification of FtsR and next generation sequencing of the enriched DNA fragments confirmed the ftsZ promoter as in vivo binding site of FtsR and revealed additional potential target genes and a DNA-binding motif. Analysis of strains expressing ftsZ under control of the gluconate-inducible gntK promoter revealed that the phenotype of the ΔftsR mutant is not solely caused by reduced ftsZ expression, but involves further targets. CONCLUSIONS: In this study, we identified and characterized FtsR as the first transcriptional regulator of FtsZ described for C. glutamicum. Both the absence and the overproduction of FtsR had severe effects on growth and cell morphology, underlining the importance of this regulatory protein. FtsR and its DNA-binding site in the promoter region of ftsZ are highly conserved in Actinobacteria, which suggests that this regulatory mechanism is also relevant for the control of cell division in related Actinobacteria.

Corynebacterium Cell Factory Design and Culture Process Optimization for Muconic Acid Biosynthesis.

Muconic acid (MA) is a valuable compound for adipic acid production, which is a precursor for the synthesis of various polymers such as plastics, coatings, and nylons. Although MA biosynthesis has been previously reported in several bacteria, the engineered strains were not satisfactory owing to low MA titers. Here, we generated an engineered Corynebacterium cell factory to produce a high titer of MA through 3-dehydroshikimate (DHS) conversion to MA, with heterologous expression of foreign protocatechuate (PCA) decarboxylase genes. To accumulate key intermediates in the MA biosynthetic pathway, aroE (shikimate dehydrogenase gene), pcaG/H (PCA dioxygenase alpha/beta subunit genes) and catB (chloromuconate cycloisomerase gene) were disrupted. To accomplish the conversion of PCA to catechol (CA), a step that is absent in Corynebacterium, a codon-optimized heterologous PCA decarboxylase gene was expressed as a single operon under the strong promoter in a aroE-pcaG/H-catB triple knock-out Corynebacterium strain. This redesigned Corynebacterium, grown in an optimized medium, produced about 38 g/L MA and 54 g/L MA in 7-L and 50-L fed-batch fermentations, respectively. These results show highest levels of MA production demonstrated in Corynebacterium, suggesting that the rational cell factory design of MA biosynthesis could be an alternative way to complement petrochemical-based chemical processes.

Identification of novel lipid modifications and intermembrane dynamics in Corynebacterium glutamicum using high-resolution mass spectrometry.

The complex cell envelopes of Corynebacterineae contribute to the virulence of pathogenic species (such as Mycobacterium tuberculosis and Corynebacterium diphtheriae) and capacity of nonpathogenic species (such as Corynebacterium glutamicum) to grow in diverse niches. The Corynebacterineae cell envelope comprises an asymmetric outer membrane that overlays the arabinogalactan-peptidoglycan complex and the inner cell membrane. Dissection of the lipid composition of the inner and outer membrane fractions is important for understanding the biogenesis of this multilaminate wall structure. Here, we have undertaken the first high-resolution analysis of C. glutamicum inner and outer membrane lipids. We identified 28 lipid (sub)classes (>233 molecular species), including new subclasses of acylated/acetylated trehalose mono/dicorynomycolic acids, using high-resolution LC/MS/MS coupled with mass spectral library searches in MS-DIAL. All lipid subclasses exhibited polarized distributions across the inner and outer membrane fractions generated by differential solvent extraction. Strikingly, deletion of the TmaT protein, which is required for transport of trehalose corynomycolates across the inner membrane, led to the accumulation of triacylglycerols in the inner membrane and to suppressed synthesis of phosphatidylglycerol and alanylated lipids. These analyses indicate unanticipated connectivity in the synthesis and/or transport of different lipid classes in C. glutamicum.

Quantitative modelling of nutrient-limited growth of bacterial colonies in microfluidic cultivation.

Nutrient gradients and limitations play a pivotal role in the life of all microbes, both in their natural habitat as well as in artificial, microfluidic systems. Spatial concentration gradients of nutrients in densely packed cell configurations may locally affect the bacterial growth leading to heterogeneous micropopulations. A detailed understanding and quantitative modelling of cellular behaviour under nutrient limitations is thus highly desirable. We use microfluidic cultivations to investigate growth and microbial behaviour of the model organism Corynebacterium glutamicum under well-controlled conditions. With a reaction-diffusion-type model, parameters are extracted from steady-state experiments with a one-dimensional nutrient gradient. Subsequently, we employ particle-based simulations with these parameters to predict the dynamical growth of a colony in two dimensions. Comparing the results of those simulations with microfluidic experiments yields excellent agreement. Our modelling approach lays the foundation for a better understanding of dynamic microbial growth processes, both in nature and in applied biotechnology.

Impact of mycolic acid deficiency on cells of Corynebacterium glutamicum ATCC13869.

Mycolic acid (MA) plays important role in Corynebacterium glutamicum, but the key enzymes in the biosynthetic pathway of MA in C. glutamicum ATCC13869 have not been characterized. Since the locus BBD29_RS14045 in C. glutamicum ATCC13869 shows high similarity to the gene Cgl2871, which encodes Pks13, the key enzyme for synthesizing MA in C. glutamicum ATCC13032, it was deleted, resulting in the mutant WG001. Compared with the wild-type ATCC13869, MA was not synthesized in WG001, but more phosphatidylglycerol and phosphatidylinositol containing longer unsaturated fatty acids were produced. WG001 cells also show hindered cell growth and defective cell separation when compared with ATCC13869 cells. Transcriptomic analysis shows that many genes relevant to the pathways of fatty acids, inositol, phospholipids, cell wall, and cell division were significantly regulated in WG001 cells when compared with ATCC13869 cells. This study demonstrates that the locus BBD29_RS14045 encodes a key enzyme that plays important role for synthesizing MA in C. glutamicum ATCC13869.

Corynebacterium glutamicum WhcD interacts with WhiA to exert a regulatory effect on cell division genes.

Corynebacterium glutamicum WhcD plays an important regulatory role in cell division. Binding of WhcD to the promoter region of its target genes, such as ftsZ, was observed by electrophoretic mobility shift assays (EMSA) using purified fusion proteins; however, binding could only be observed in the presence of WhiA. Although WhcD alone did not bind to the DNA, it stimulated binding of WhiA to the promoter region of the cell division gene ftsZ. Binding of WhcD and WhiA to DNA did not occur in the presence of the oxidant diamide. Purified WhcD and WhiA physically interacted in vitro. The presence of diamide did not disrupt the WhcD-WhiA interaction but affected binding of WhiA to the promoter region of ftsZ. The GACAC motif and adjacent sequences were found to be important for binding of the WhcD-WhiA complex to the DNA. Collectively, our results suggest that WhcD enhances the WhiA DNA-binding activity by physically interacting with WhiA. In addition, loss of WhiA DNA-binding activity in the presence of an oxidant agent may suggest a role for this protein as a switch that controls cell division in cells under oxidative stress.

Heat-killed cell preparation of Corynebacterium glutamicum stimulates the immune activity and improves survival of mice against enterohemorrhagic Escherichia coli.

Fermentation by Corynebacterium glutamicum is used by various industries to produce L-Glutamate, and the heat-killed cell preparation of this bacterium (HCCG) is a by-product of the fermentation process. In present study, we evaluated the immunostimulating and survival effects against enterohemorrhagic Escherichia coli (STEC) infection of HCCG. HCCG significantly stimulated in vitro IgA and interleukin-12 p70 production in murine Peyer's patch cells and peritoneal macrophages, respectively. Oral administration of 10 mg/kg body weight (BW) of HCCG for seven consecutive days stimulated IgA concentration in murine cecal digesta. Mice were orally administered HCCG for 17 consecutive days (d0-d17), and challenged with STEC on d4 to d6. Survival of mice tended to improve by 100 mg/kg BW of HCCG administration compared with those in control group. In conclusion, HCCG supplementation was found to prevent STEC infection in mice, and thus it may have the potential to stimulate the immune status of mammals.

The whcD gene of Corynebacterium glutamicum plays roles in cell division and envelope formation.

In this study, we analysed the whcD gene from Corynebacteriumglutamicum, which encodes a homologue of whiB, a Streptomycescoelicolor gene required for the sporulation of aerial hyphae. Deletion of the gene (ΔwhcD) severely affected cell growth in C. glutamicum. The ΔwhcD strain exhibited a large filamentous, branched and bud-shaped morphology with multiple septa. The transcription levels of the cell division genes involved in Z-ring assembly and septal peptidoglycan synthesis, including ftsZ, sepF, ftsQ and ftsI, were markedly decreased in the ΔwhcD strain. The divIVA gene, which is responsible for apical growth, also showed decreased transcription in the ΔwhcD strain. However, genes involved in the later stages of cell division, such as cell separation and chromosome segregation, did not show notable changes in their transcription levels. Moreover, the mutant strain was susceptible to inhibitors of transpeptidation, including penicillin and vancomycin. In addition, the transcription of genes fas-IA, fas-IB and accD1, which participate in the synthesis of fatty acid and cell envelope component mycolic acid, was altered in the ΔwhcD strain. This increased the cell surface hydrophobicity in the mutant strain, apparently leading to cell aggregation in liquid media. These findings indicate that whcD is a whiB-like gene with roles in the early stages of cell division and fatty acid synthesis, and the pleiotropic phenotypes of the ΔwhcD strain suggest that whcD may be a global regulatory gene.

Metabolic engineering of Corynebacterium glutamicum for shikimate overproduction by growth-arrested cell reaction.

Corynebacterium glutamicum with the ability to simultaneously utilize glucose/pentose mixed sugars was metabolically engineered to overproduce shikimate, a valuable hydroaromatic compound used as a starting material for the synthesis of the anti-influenza drug oseltamivir. To achieve this, the shikimate kinase and other potential metabolic activities for the consumption of shikimate and its precursor dehydroshikimate were inactivated. Carbon flux toward shikimate synthesis was enhanced by overexpression of genes for the shikimate pathway and the non-oxidative pentose phosphate pathway. Subsequently, to improve the availability of the key aromatics precursor phosphoenolpyruvate (PEP) toward shikimate synthesis, the PEP: sugar phosphotransferase system (PTS) was inactivated and an endogenous myo-inositol transporter IolT1 and glucokinases were overexpressed. Unexpectedly, the resultant non-PTS strain accumulated 1,3-dihydroxyacetone (DHA) and glycerol as major byproducts. This observation and metabolome analysis identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-catalyzed reaction as a limiting step in glycolysis. Consistently, overexpression of GAPDH significantly stimulated both glucose consumption and shikimate production. Blockage of the DHA synthesis further improved shikimate yield. We applied an aerobic, growth-arrested and high-density cell reaction to the shikimate production by the resulting strain and notably achieved the highest shikimate titer (141g/l) and a yield (51% (mol/mol)) from glucose reported to date after 48h in minimal medium lacking nutrients required for cell growth. Moreover, comparable shikimate productivity could be attained through simultaneous utilization of glucose, xylose, and arabinose, enabling efficient shikimate production from lignocellulosic feedstocks. These findings demonstrate that C. glutamicum has significant potential for the production of shikimate and derived aromatic compounds.

Corynebacterium glutamicum MTCC 2745 immobilized on granular activated carbon/MnFe2O4 composite: A novel biosorbent for removal of As(III) and As(V) ions.

The optimization of biosorption/bioaccumulation process of both As(III) and As(V) has been investigated by using the biosorbent; biofilm of Corynebacterium glutamicum MTCC 2745 supported on granular activated carbon/MnFe2O4 composite (MGAC). The presence of functional groups on the cell wall surface of the biomass that may interact with the metal ions was proved by FT-IR. To determine the most appropriate correlation for the equilibrium curves employing the procedure of the non-linear regression for curve fitting analysis, isotherm studies were performed for As(III) and As(V) using 30 isotherm models. The pattern of biosorption/bioaccumulation fitted well with Vieth-Sladek isotherm model for As(III) and Brouers-Sotolongo and Fritz-Schlunder-V isotherm models for As(V). The maximum biosorption/bioaccumulation capacity estimated using Langmuir model were 2584.668mg/g for As(III) and 2651.675mg/g for As(V) at 30°C temperature and 220min contact time. The results showed that As(III) and As(V) removal was strongly pH-dependent with an optimum pH value of 7.0. D-R isotherm studies specified that ion exchange might play a prominent role.

Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE.

Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass.

RNase III mediated cleavage of the coding region of mraZ mRNA is required for efficient cell division in Corynebacterium glutamicum.

The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3'-to-5' exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596.

Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform.

Cell-to-cell heterogeneity typically evolves due to a manifold of biological and environmental factors and special phenotypes are often relevant for the fate of the whole population but challenging to detect during conventional analysis. We demonstrate a microfluidic single-cell cultivation platform that incorporates several hundred growth chambers, in which isogenic bacteria microcolonies growing in cell monolayers are tracked by automated time-lapse microscopy with spatiotemporal resolution. The device was not explicitly developed for a specific organism, but has a very generic configuration suitable for various different microbial organisms. In the present study, we analyzed Corynebacterium glutamicum microcolonies, thereby generating complete lineage trees and detailed single-cell data on division behavior and morphology in order to demonstrate the platform's overall capabilities. Furthermore, the occurrence of spontaneously induced stress in individual C. glutamicum cells was investigated by analyzing strains with genetically encoded reporter systems and optically visualizing SOS response. The experiments revealed spontaneous SOS induction in the absence of any external trigger comparable to results obtained by flow cytometry (FC) analyzing cell samples from conventional shake flask cultivation. Our microfluidic setup delivers detailed single-cell data with spatial and temporal resolution; complementary information to conventional FC results.

Modeling and CFD simulation of nutrient distribution in picoliter bioreactors for bacterial growth studies on single-cell level.

A microfluidic device for microbial single-cell cultivation of bacteria was modeled and simulated using COMSOL Multiphysics. The liquid velocity field and the mass transfer within the supply channels and cultivation chambers were calculated to gain insight in the distribution of supplied nutrients and metabolic products secreted by the cultivated bacteria. The goal was to identify potential substrate limitations or product accumulations within the cultivation device. The metabolic uptake and production rates, colony size, and growth medium composition were varied covering a wide range of operating conditions. Simulations with glucose as substrate did not show limitations within the typically used concentration range, but for alternative substrates limitations could not be ruled out. This lays the foundation for further studies and the optimization of existing picoliter bioreactor systems.